An EPO‐loaded multifunctional hydrogel synergizing with adipose‐derived stem cells restores neurogenic erectile function via enhancing nerve regeneration and penile rehabilitation

Abstract Neurogenic erectile dysfunction (nED) is one of the most common and intractable postoperative complications of rectal and prostate cancer surgery and sometimes accompanies patients lifelong. The transplantation of stem cells has been proved a promising way for treatment. However, the therapeutic efficacy is severely impaired by excessive cell loss and death and poor accumulation in the injury site along with the traditional implantation strategy. Herein, an EPO‐loaded multifunctional hydrogel was designed. The hydrogels' adhesive property and mechanical strength were enhanced by adding catechol‐catechol adducts, thus significantly improving adipose‐derived stem cells (ADSC) retention and rescuing cell loss in the injury site. Meanwhile, the sustained release of EPO effectively ameliorated the viability and paracrine activity of ADSC, leading to enhanced migration of Schwann cells and differentiation of PC12 cells in vivo. On a bilateral cavernous nerve injury rat model, the present stem cell‐EPO‐hydrogel implanted strategy could significantly alleviate erectile dysfunction. The higher expression of Tuj1 and lower expression of GFAP in the major pelvic ganglia (MPG) indicated the acceleration of neural differentiation while the suppressing development of astrocytes. Also, the combined therapy restored the expression levels of eNOs, nNOs, and α‐SMA in penile tissues, suggesting the rehabilitation of the penis. Further analysis of Masson trichrome staining and apoptosis evaluation of the corpus cavernosum showed the preservation of vascular endothelium content and the prevention of penile fibrosis after denervation. Overall, we believe that this combined strategy presents a promising way not only for restoring neurogenic erectile function but also for the clinical translation of stem cell therapy.


| INTRODUCTION
Rectal and prostate cancers are both among the most common malignant tumors with increasing prevalence worldwide. Radical resection of the lesion is one of the most efficient treatment options. Postoperative erectile dysfunction (ED) is a significant complication and frequently occurs in male patients for the sake of inadvertent crush, tension, and resection injuries to the pelvic autonomic nerve (PAN) during the surgical procedure. 1,2 Although many advances have been made, such as more elaborate surgical anatomy and nerve-sparing techniques, the incidence of neurogenic erectile dysfunction (nED) remains high. 3,4 Furthermore, phosphodiesterase-5 inhibitors, which are the most popular choice for ED patients, have encountered significant challenges in the treatment of nED. 5 Besides, the therapeutic effect of alternatives, including the intracavernous injections (ICs) of alprostadil or prosthetic implants, is too limited to cure nED. It is therefore highly imperative to explore new therapeutic strategies to improve clinical outcomes in these cases. 6 Unlike ED resulting from aging and diabetes, nED is essentially a neurotraumatic disease. Previous studies have revealed that sufficient penile erection cannot be imagined without a sound neural network, an intact vascular system, and healthy cavernosal tissue. 7 The major pelvic ganglion (MPG), cavernous nerve (CN), and dorsal penile nerve are the primary nerves that innervate the penis. Once an injury occurs, nerve impulse conduction is affected, and the connections between MPG/CN and penis are interrupted. In turn, denervation will induce the downstream remodeling in the penile tissues, such as the apoptosis of smooth muscle, fibrosis of the corpus cavernosum, and loss of endothelial content, resulting in inevitable nED. 8,9 As a result, optimal management of patients with nED should include timely and effective nerve regeneration and penile rehabilitation.
In the past decades, emerging evidence has proved that stem cell therapy such as adipose-derived stem cells (ADSC) holds great potential for nerve regeneration and tissue engineering. 10 Kim et al. 11 reported an ADSC-laden nanofibril composites to reconstruct cartilage and subchondral bone ECM matrices. Leong et al. 12 prepared surface tethering of inflammation modulatory nanostimulators to ADSC for ischemic muscle repair. Yan et al. 13 concluded that ADSC overexpressing N-cadherin enhanced angiogenesis and cardiomyocyte proliferation after ischemic heart injury. The underlying therapeutic mechanism may be the differentiation of stem cells into specialized cell types (multipotency) 14 or paracrine effects 15,16 that induce immune responses and stimulate revascularization and angiogenesis. Nevertheless, a clinical trial conducted by Bahk et al. 17 demonstrated that the rigidity had increased in the penis while it was insufficient for penetration after infusion of stem cells into the corpus cavernosum. In addition, Fang et al. 18 concluded that the red signal of PKH-26-labeled stem cells was only detectable in the first 2 days after periprostatic implantation (IP). It could be implied that the therapeutic efficacy was severely compromised by excessive cell loss and death and poor accumulation in the injury site accompanying traditional implantation strategy. 16,19 Hence, more advanced technologies urgently need to enhance stem cell viability for better treatment.
Hydrogels are polymeric networks that bond together by physical and/or covalent crosslinks. Due to its excellent biological properties, biodegradability, and biocompatibility, hydrogels derived from natural biomaterials have been extensively developed in tissue engineering and nerve regeneration. Meanwhile, increasing evidence has demonstrated the advantage of the combined implantation of stem cells with biocompatible hydrogels over utilizing alone, in which hydrogels provide a temporary shelter for cell adhesion, proliferation, and migration, while the implanted cells modulate the local microenvironment to compensate for the damaged tissues. 10,14,20 For example, Li et al. 14 fabricated a multifunctional hydrogel encapsulating mesenchymal stem cells (MSC) to induce neural differentiation, contributing to significant nerve regeneration in a spinal cord injury model.
Besides, due to the intricacy of the microenvironment after nerve injury such as Wallerian degeneration and inflammatory infiltration, the addition of exogenous of neuro-protective-related cytokines has been revealed to further improve stem cell therapeutic efficiencies, such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), brain-derived neurotrophic factor (BDNF), NT3, and NT4. [21][22][23][24] As a renal hormone regulating hematopoiesis, erythropoietin (EPO) is initially applied for anemia recovery.
Recently, accumulating evidence suggests its neuroprotective and neurotrophic activities mediated by EPO-receptor (EPOR) expressed by neurons in the nervous system. 25,26 Moreover, EPO is revealed to improve the migration of Schwann cells (SCs) in the injured nerve via the local production of fibronectin. 27 Pretreatment or modification by EPO was reported to enhance the proliferation and migration of stem cells, thus promoting its protective effects in nerve injury. 28,29 Meanwhile, EPO receptor expression was identified and localized in penile tissues and the periprostatic neurovascular bundles responsible for erectile function. 30 Herein, an EPO-loaded multifunctional hydrogel was constructed for cell implantation to an nED rat model (Scheme 1). Methacrylate gelatin (GelMA) was chosen as the main matrix material for its excellent biocompatibility to simulate the biological function of the natural extracellular matrix (ECM). Meanwhile, the containing RGD motifs could interact with integrins to enhance cell adhesion. 31,32 In addition, mussel-inspired chemistry was applied to compensate for the insufficient viscous properties of GelMA. 33 Dopamine-modified hyaluronic acid (HADA) and carboxymethyl chitosan (CMCDA) were further introduced to confer enhanced mechanical strength to the systems, ensuring efficient cell retention in the nerve injury site even if rats were highly active. Moreover, the sustained release of EPO improved the viability of ADSC and synergistically promoted the migration of SCs, contributing to highly efficient nerve regeneration and penile rehabilitation. On a bilateral cavernous nerve injury rat model (BCNI), the hydrogel system elicited significant restoration of erectile function. As a step toward clinical applications, the present stem cell-EPOhydrogel implanted strategy provides a promising method to realize the stem cell therapies of nED.

| Reagents
Gelatin type A (from porcine skin), methacrylic anhydride (MA), and carboxymethyl chitosan (CMC) were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Active erythropoietin (EPO) was bought from Cloud-Clone Crop. Hyaluronic acid (HA) was obtained from Freda Biopharma Co., Ltd. Dopamine hydrochloride (DA, purify >98%) was bought from Aladdin Biochemical Technology Co., Ltd. All reagents were of analytical grade unless otherwise noted.

| Preparation of GelMA, HADA, and CMCDA hydrogels
GelMA was prepared as previously described with slight modifications. 32 Briefly, 10 g gelatin power was wholly dissolved in 100 ml PBS at 50 C, then added dropwise methacrylic anhydride (MA) at a mass ratio of 0.6:1. After reaction for at least 6 h under constant agitation at 50 C, the obtained solution was processed to dialyze against deionized water for 3 days to remove all the unreacted reagents (MWCO:12-14 kDa). Finally, the GelMA solution was lyophilized at À80 C and stored at À20 C before use.
HADA was synthesized by an EDC/NHS reaction. 33 Generally, 1 g HA was utterly dissolved in deionized water (100 ml) under a nitrogen atmosphere, followed by the supplement of 575-mg EDC and 345 mg NHS. The mixture was stirred for 20 min before adding 569 mg DA. 0.1 M NaOH or HCl was utilized to adjust the pH value of the mixture between 5 and 6. After 12-h reaction at room temperature, the solution was dialyzed for 3 days (MWCO: 1 kDa) to remove all impurities under acidic conditions. The final HADA solution was lyophilized at À80 C and stored at À20 C for further use.
To prepare the CMCDA conjugate, 1-g CMC was first completely dissolved in deionized water (20 ml) at 60 C, followed by the addition of 575-mg EDC. In addition, 345 mg NHS and 569 mg DA were weighed and dissolved in 20 ml deionized water. Then, the CMC/EDC solution was dropwise added to the DA/NHS mixture. The pH value S C H E M E 1 Concept illustration. GelMA is fabricated and the adhesive property is improved via DA polymerization. Then, the EPO-loaded hydrogel synergizing with ADSC is applied to restore neurogenic erectile function induced by crush injury to bilateral cavernous nerve via enhancing nerve regeneration and penile rehabilitation was kept at 5, and the by-products were removed by centrifugation after reaction in nitrogen ambient for 6 h. The supernatant was harvested and dialyzed against hydrochloric acid (pH = 5) for 24 h, then dialyzed against deionized water. The CMCDA solution was lyophilized at À80 C and stored at À20 C before use.
A 365-nm UV laser was applied for gelatinization.

| Swelling ability
A conventional gravimetric method was performed to assess the swelling properties. Lyophilized prepared hydrogels were weighed and recorded as m 0 . Then, the hydrogels were submerged in PBS at 37 C, and the swollen weight was measured at selected time intervals and recorded as m 1 where w 0 and w 1 represent the dry weight of the initial hydrogels and the remaining hydrogels at different time point, respectively. SCs were isolated from the sciatic nerve of 2-week-old SD rats. First, the rats were killed, and the bilateral sciatic nerves were collected. Then, the nerve bundle was cut into ≈3-mm segments and incubated in high-glucose DMEM supplemented with 10%

| EPO integration and in vitro release behavior of EPO
FBS at 37 C in a humidified atmosphere containing 5% CO2. Five micrograms per milliliter of cytarabine was used to purify SC. Immunofluorescence staining for S100β was performed for the identification of SC.
PC12 cells (rat adrenal pheochromocytoma-derived cell line) were obtained from Zhongqiao Xinzhou Biotechnology Co., Ltd., and cultured in DMEM supplemented with 10% FBS for proliferation.

| Biocompatibility test in vitro
Calcein-AM/PI double staining, cytoskeleton staining, and cell cou- Additionally, a CCK-8 assay was performed to assess the cell viability according to the protocol. The absorbance value was measured at λ = 450 nm.

| ADSC encapsulation, viability, and spreading
ADSC encapsulation into the hydrogels was performed on previously published protocols with slight modifications. 35

| Enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) was performed to investigate the paracrine response of ADSC to hydrogels. Briefly, 100-μl pre-gels with or without EPO were added to each well of the 24-well plate and gelated before the experiment, and the well without any supplement was set as control. Then, ADSC was seeded into the 24-well plate at the density of 1 Â 10 4 cells/well and cultured in a fresh medium at 37 C. After co-culture for 3 days, the supernatant of each well was harvested, and the level of neuro-protective related cytokines (VEGF, BDNF, NGF, and PDGFα) was determined by ELISA kits (Meimian Biotechnology Co. Ltd.). Three independent experiments were conducted for statistical analysis. pared and anesthetized with 2.5%-3% isoflurane. Next, the MPG/CN were exposed posterolaterally on both sides of the prostate. 37 The bilateral CN were crushed 5 mm away from MPG as previously described. 34,37 After that, rats were randomly divided into five groups with different treatments (n

| Erectile function evaluation
Four weeks after surgery, both intracavernous pressure (ICP) and mean arterial blood pressure (MAP) were recorded continuously to evaluate the erectile function of animals. After anaesthetization, the right carotid artery was exposed through a midline incision from the

| Western blot analysis
Relative expression levels of the expected proteins in penile tissues were detected by western blot analysis as previously described. 34  Notably, the characteristic peak of the amide bond (V C═O ) was not observed in the spectrum of CMCDA, which was considered to be masked by the C═C peak. For UV-vis absorption spectra, a slight blue shift (≈4 nm) was observed both in HADA and CMCDA, confirming the generation of amide linkages. Figure S1a showed the gelation process of GelMA/HADA/CMCDA hydrogels triggered by UV light. Figure S1b displayed the appearances of hydrogels with different components. Besides, the SEM results ( Figure S1c) confirmed the interconnected and homogeneous pore structures of all hydrogels. In conclusion, these results confirmed the successful fabrication of polymers for further hydrogel preparation.

| Physical and mechanical properties of hydrogels
The swelling ability was related to the porosity and the cross-linking degree. Hydrogels with certain swelling abilities could prove good mechanical strength and water absorption for implanted cells for proliferation and nutrition. 39 As shown in Figure 1d, hydrogels absorbed the PBS rapidly and reached their equilibrium after 7 h. Apparently, the supplement of HADA and/or CMCDA had improved the swelling performance of GelMA, which was attributed to the sponge-like structure of CMC and the good water suction property of HA. The swelling ratio of GelMA and GelMA/HADA/CMCDA was 35.5% and 60.3%, respectively. Additionally, a decrease in swelling ratio was observed when HADA was added to GelMA/CMCDA, which might be ascribed to the enhanced crosslinking density.
We assumed that the introduction of HADA and CMCDA could significantly improve the mechanical strength of hydrogels for further implantation based on GelMA. To test this hypothesis, rheological analysis was first performed. As revealed in Figure 1e, the elastic modulus (G 0 ) was significantly higher than the viscous modulus (G 00 ), with shear frequency varying from 0.1 to 10 Hz among groups, indicating the solid elastic properties. As shown in Figure 1f, the time-sweep test was also performed. Only a mild reduction was observed both in G 0 and G 00 . Meanwhile, G 0 was consistently higher than G 00 throughout the experiment (600 s). The results suggested that hydrogels could maintain their structurally stable 3D network. Notably, GelMA/ HADA/CMCDA demonstrated the lowest G 0 , indicating the most resilient performance.
Next, the adhesive properties and compression strength of hydrogels were evaluated. For adhesive properties assessment, hydrogels were applied between two pieces of fresh porcine skins and the lap shear tests were performed. 40 As shown in Figure 1g

| In vitro degradation assessment
As an implant material, it is essential for hydrogels to be biodegradable. Therefore, the degradation behavior of hydrogels was observed by immersing hydrogels in PBS. Lysozyme (1000 U/ml) was used to simulate the in vivo environment. 41 The results are displayed in Figure S2. Obviously, the degradation rate of hydrogels was slower without lysozyme. It took about 14 days for hydrogels to degrade completely without lysozyme, while only about 7 days for hydrogels with lysozyme ( Figure S2a,b). Additionally, compared with pure GelMA, hydrogels supplemented with HADA and/or CMCDA degraded faster in the first few days, since there was no chemical cross-linking network between HADA/CMCDA and GelMA. SEM was also utilized to observe the changes in the microstructure of hydrogels ( Figure S2c,d). Apparently, under the enzyme condition, the disintegration rate of the cavity in the hydrogel materials was faster than those without enzyme, which was consistent with the degradation profiles.

| Biocompatibility of the hydrogels with ADSC
Compared with other kinds of stem cells, ADSC is easy to isolate from tissues and possesses the properties of higher activity to secrete neuroprotective related cytokines and immunoregulatory factors while lower immunogenicity. 34,42 As shown in Figure S3a1 ECM components to enhance cell adhesion, viability, and proliferation. 31,33,38 Herein, the prepared hydrogel was an ideal carrier for ADSC encapsulation and in vivo implantation.

| The effects of hydrogels on the SC migration
Peripheral nerve regeneration is a complex biological process, and that immediately occurs after nerve injury. SCs are the primary cells that make up the peripheral nervous system, which is activated to phagocytize the debris in the initial time and enhance neurite growth by producing neuro-protective related factors in the later period. 8,45 Considering the critical role of SC in nerve regeneration, we evaluated the effects of different hydrogel systems on cell migration. Primary SC isolated from the sciatic nerve were immunofluorescent identified by S100β 46 ( Figure S5a). Next, SC was incubated in the upper chambers.
After being cultured for 48 h, the numbers of cells penetrating the lower chambers were evaluated. Figure 3a illustrated the representative results of each group, and Figure S5b was the semi-quantitative analysis results. Obviously, the incubation of SC in ADSC supernatants led to more cell migration than the control group, and the chemotaxis effect was enhanced by seeding ADSC on hydrogels with/ without the supplement of EPO. Interestingly, the supernatant of Gel/EPO alone could lead to certain numbers of cells penetrating the lower chambers, which was related to the effect of EPO on cellular migration. 28,47 To investigate the underlying mechanism of enhanced migration, the secretion levels of neuro-protective related factors in supernatants were detected. As shown in Figure S6, the ADSC cultured on EPO-loaded hydrogels showed the highest levels of GDNF, BDNF, NGF, and PDGFα proteins secretion, indicating the enhanced paracrine activity.

| Neurite growth assessment on PC12 cells
To verify the capability of the hydrogel complex in nerve regeneration, PC12 cells were incubated with the supernatants, and the morphology was observed to evaluate the effects of hydrogels on cell differentiation ( Figure 3c). As shown in Figure 3b, undifferentiated PC12 cells presented the common spindle-shaped or polygonal shapes. Figure 3d illustrates the representation images after incubation for 1 day and 3 days. Compared with the control group, cells cultured in supernatants presented typical nerve cell morphology, with some multipolar cells with longer synapses. We further divided differentiated PC12 cells into five groups (from L0 to L4) to quantify the elongation of neural processes 36 (Figure 3e,f). Enhanced cell differentiation was observed even within a 1-day treatment. The percentage of L0 was about 60% in the control group versus 30% in Gel/EPO/ ADSC group. At the end of the third day, the percentage of L0 in control groups remained about 47%, while the rate decreased to about 17% in Gel/EPO/ADSC group, with an increased percentage of highly differentiated cells (from L2 to L4). Additionally, PC12 cells in other treatment groups also showed varying degrees of differentiation, suggesting that the capacity in nerve repair of ADSC was enhanced by culturing on hydrogels. Meanwhile, the cell differentiation appeared to be mildly improved compared with the control group, which was ascribed to the increased mitochondrial activity and the attenuation of oxidative stress effect of EPO on PC12 cells. 48 Together, these results strongly verified the potential of stem cell-EPO-hydrogel complex in the promotion of neurite outgrowth.  (Figure 4b-d).

| Erectile function restoration in BCNI rats
The strategy of EPO-loaded hydrogel could significantly enhance the therapeutic effect of ADSC (p < 0.001). In addition, EPO receptor expression was identified and localized in penile tissues and the periprostatic neurovascular bundles responsible for erectile function. 30 Allaf et al. 49 reported that exogenous administration of 5000 IU/kg in the setting of BCNI promoted erectile function recovery. In our study, the ICP max /MAP value and relative total ICP/MAP value of Gel and Gel/EPO group were higher than those of the PBS group, suggesting the benefits of hydrogel materials and EPO in function restoration.

| Cell tracing
At present, stem cells have been preliminarily explored in nED treatment research, of which the administration routes are IC and IP. 50,51 It has been reported that the route of IC focuses on nerve regeneration, while that of IP attenuates the degeneration of penile tissues caused by neuronal Wallerian degeneration. 52 Although the two approaches showed similar therapeutic effects in short-term treatment (2 or 4 weeks), as a neurotraumatic disease, giving priority to rapid repair of damaged nerves is the best choice to deal with nED.
Fandel et al. 37  One-way ANOVA followed by post hoc Bonferroni analysis. *p < 0.05 and ***p < 0.001. Note that Gel represents GelMA/HADA/CMCDA hydrogel although cell-fibrin scaffolds were usually adopted to encapsulate stem cells in the administration route of IC, the therapeutic efficacy was severely compromised by the poor accumulation in the injury site to function effectively. It, therefore, seems that the implantation of abundant cells with high viability could lead to improved performance of stem cells in the restoration of erectile function. In this study, we proposed an innovative method for accurate delivery of ADSC to the injury site via an adhesive hydrogel. First, we assessed the adhesive property of hydrogels in vivo. After treatment with hydrogels for 1, 3, and 7 days, rats were anesthetized and dissected. As shown in Figure S7a,

| Rehabilitation of target penile tissues
A good neural network, an intact vascular system, and healthy cavernosal tissue ensure sufficient penile erection. Damage to CN will lead to a series of pathophysiological changes in the architecture of the penis, such as loss of endothelial tissues, denervation, and decrease of cavernosal smooth muscle ingredient. 53,54 Moreover, these changes were reported to continue to progress in the later period, even if CN was successfully repaired by that time. 55 To further observe the rehabilitation of the penile tissues elicited by different treatments, immunofluorescent staining of penis transverse sections was performed. Endothelial tissue is the guarantee of the sufficient engorgement of the penis during erections, of which the recognized marker is eNOS. 56 As shown in Figure 6a Taken together, these results showed consistent outcomes with erectile function detection and nerve regeneration assessment, suggesting that the EPO-loaded hydrogel synergizing with ADSC contributed to restoring nED via enhancing rapid nerve regeneration and penile rehabilitation.

| Apoptosis evaluation
Nerve injury can lead to different degrees of dysfunction of target organs. Although the damage is limited to the neuron, the functional recovery involves the neuronal cell body, axon, and target organ.
Therefore, the study of nerve injury mainly includes three aspects: the protection of neurons, the promotion of nerve regeneration, and the reported to occur as early as 1 week after BCNI. 34,54 It is therefore necessary to repair the nerve quickly and effectively in the early stage.
In this study, the appropriate degradation rate and adhesive properties of hydrogels, the sustained release of EPO, and the enhanced viability of ADSC were well satisfied with the critical time window of nED treatment. Herein, to assess whether the curative effect of nerve regeneration could alleviate apoptosis and fibrosis of penile tissues (target organ), Masson trichrome staining of the corpus cavernosum was first performed. Figure 7a shown the representative results of each group, in which smooth muscle was stained red while F I G U R E 7 ADSC-EPO-hydrogel combing strategy preserved vascular endothelium content and inhibited penile fibrosis via exerting antiapoptotic effects. (a) Masson's trichrome staining of the penile and the smooth muscle/collagen ratio (c) among groups. Fluorescent immunostaining (b) and relative fluorescence intensity of Caspase-3 (d) among groups. (e) Caspase-3, Bcl-2, and BAX protein expression in penile tissues. One-way ANOVA followed by post hoc Bonferroni analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. Note that Gel represents GelMA/ HADA/CMCDA hydrogel connective tissue was stained blue, respectively. Obviously, BCNI led to severe atrophy of cavernosa smooth muscle, with the lowest ratio value of smooth muscle to collagen (PBS group). Meanwhile, treatment with ADSC could restore the smooth muscle ingredient in penile, of which the curative effect was enhanced by the EPO-loaded hydrogel implanted strategy (Figure 7c). Similar curative results were observed in immunofluorescent staining of caspase-3 on penile tissues (Figure 7b,d). In addition, the expression levels of apoptosisrelated proteins (caspase-3, Bcl-2, and BAX) in penile tissues was explored via western blot analysis (Figure 7e). After an injury to MPG/CN, the expression levels of caspase-3 and BAX were increased while that of Bcl-2 was decreased, indicating the process of apoptosis.
The implantation of ADSC could attenuate these effects. Thus, it could be inferred that the stem cell-EPO-hydrogel implanted strategy could prevent the atrophy of the penis via inhibiting apoptosis and the fibrosis process.

| CONCLUSION
In this study, we designed and fabricated an EPO-loaded

CONFLICT OF INTERESTS
The authors declare no conflict of interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.